Supercoiled DNA promotes formation of intercalated cis-N2-deoxyguanine adducts and base-stacked trans-N2-deoxyguanine adducts by (+)-7R,8S-dihydrodiol-9S,10R-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene

The highly reactive and mutagenic benzo[a]pyrene metabolite, (+)-7R,8S-dihydroxy-9S,10R-epoxy- 7,8,9, 10-tetrahydrobenzo [a] pyrene (BPDE), forms predominantly N-2-deoxyguanine DNA adducts in two stereoisomeric configurations (cis and trans). In previous in vitro assays using oligonucleotide substrates site specifically modified with cis- and trans-BPDE adducts, the nucleotide excision repair (NER) systems of eukaryotes and prokaryotes incise cis-BPDE adducts more efficiently than trans-BPDE adducts [Hess, et al. (1997) Mol. Cell Blol 17, 7069; Zou, et al. (2001) Biochemistry 40, 2923). We investigated the influence of DNA secondary structure on stereospecificity of BPDE adduct formation, and incision of BPDE adducts by the prokaryotic UvrABC NER endonuclease was examined. BPDE adducts formed at low density on supercoiled plasmids were incised 6-7-fold better by the thermoresistant Bacillus caldotenax UvrABC than were BPDE adducts formed on linear DNA. Linearizing supercoiled plasmid DNAs after BPDE adduct formation did not diminish incision efficiency. These results suggested that configuration and/or conformation of adducts formed on linear and supercoiled DNAs differed. This hypothesis was confirmed by low temperature fluorescence spectroscopy of adducted supercoiled and linear DNAs. Spectroscopic results indicated that intercalated cis-BPDE adducts as well as base-stacked trans-BPDE adducts formed more abundantly in supercoiled DNA than in linear DNA. A higher cis to trans adduct ratio in supercoiled DNA was confirmed by high resolution [P-32]postlabeling analyses. These results demonstrate that DNA secondary structure influences both configuration and conformation of BPDE adducts formed at low density (similar to1 adduct/kbp) and suggests that the ratio of cis- to trans-BPDE adducts and amount of base-stacked trans adducts formed under physiological exposure conditions may be higher than inferred from high dose experiments.